The proposed studies will focus on the signaling cascade responsible for the estrogen- and shear stress-specific regulation of cytochrome P450 (CYP) gene and protein expression, leading to an enhanced release of EETs to dilate/hyperpolarize vessels that are deficient in NO. Two specific aims are proposed related to 1) estrogen specific pathways and 2) shear stress- dependent pathways. Studies will be conducted on isolated arteries of endothelial nitric oxide knockout (eNOS-KO) and estrogen receptor (ER)-KO mice. Specific CYP family/subfamily gene expression, protein synthesis and EET regioisomers responsible for EET-mediation of flow-induced dilation/hyperpolarization in vessels that have been exposed to estrogen (Specific Aim 1) or shear stress (Specific Aim 2) will be assessed by using microarray/real time RT-PCR, molecular (western blotting and immunohistochemistry) and fluorescence HPLC analyses. The specific target for EETs will be determined by patch-clamp techniques. By using a newly designed vessel culture technique, an RNA interference (RNAi) study will be performed to confirm the functional significance of the gene(s)/protein(s)/product(s) detected in the vessels, by comparison of flow-induced dilation/hyperpolarization in intact vessels before and after transfection of the vessels with specific siRNA. Moreover, roles of tyrosine kinases/MAPK pathway, as one of the most characterized extranuclear signaling actions of estrogen in the regulation of CYP expression and its dowmstream located specific transcription factor(s) will also be assessed. In addition, as a consequence of activation of CYP, the impact of superoxide/hydrogen peroxide on estrogen- and shear stress-specific signaling cascades will be evaluated. The interactions among the two pathways will be clarified by comparison of the responses of vessels treated with a single stimulus (e.g. estrogen, or shear stress) with those treated with combined stimuli (e.g. estrogen plus shear stress). Also, the specific role of ERs in the estrogen-specific pathway involving the regulation of CYP will be assessed by comparison of the responses observed from vessels that are absent in ER1 or ER2 with those of wild type controls. The knowledge generated by these studies will unravel a novel therapeutic target to improve vascular endothelial function by the upregulation of CYP-dependent dilator pathways as an adaptation to NO deficiency. PUBLIC HEALTH RELEVANCE: The endothelium is the inner layer of blood vessels. Endothelial cells release vasoactive factors (agents) when they are stimulated by estrogen or shear stress. Estrogen(s) is a female hormone(s) that is primarily produced by the female reproductive system, such as the ovaries. Shear stress is the mechanical force on the endothelium when blood flows through the vessels. The factors released from endothelial cells can dilate (enlarge) the vessels, leading to greater blood supply to organs/tissues. Thus, this project is to study how endothelial cells release the agents when stimulated with estrogen and shear stress.